During the course of preliminary experiments concerning the metabolic transformations of tritiated pyridoxine (PN) in animals with transplantable hepatomas we observed a novel labeled vitamin B6 metabolite peculiar to hepatomas. In these tests, uniformly labeled PN was administered intraperitoneally to control and tumor-bearing rats and the metabolic transformations of the vitamin were studied in liver and the hepatomas. Rats were killed at different time intervals following label injection, livers and tumors removed and vitamin metabolites isolated from homogenates using acid extraction. The labeled metabolites were chromatographically separated on Dowex-H(plus) ion exchange columns using linear pH gradient elution. Vitamin B6 standard compounds were also similarly chromatographed individually and also as mixtures. Further identification of each eluting peak was made using high voltage electrophoresis (Savant) and thin layer chromatography on silica gel (BBA 451, 333-341, 1976). The occurrence of a novel, radioactively labeled peak was observed only when extracts from hepatomas were chromatographed on Dowex columns. The labeled peak eluted before pyridoxal and after elution of the three phosphorylated vitamin B6 forms. Further thin layer chromatography on silica gel showed that this novel labeled vitamin metabolite was not pyridoxic acid or any of the known vitamin B6 compounds (i.e., pyridoxine, pyridoxal, pyridoxamine and their phosphorylated derivatives). For this application I propose experiments to identify and characterize this novel vitamin B6 metabolite and assess its importance to the economy of the tumor cell. The proposed studies may well show a new vitamin B6 metabolic pathway in tumor cells which may be mediated by an as yet unknown enzyme protein which would be characterized.